RNA isolation

Orac Orac_USA at hotmail.com
Fri Nov 19 11:59:37 EST 1999


In article <0a0133f8.fbacd721 at usw-ex0107-043.remarq.com>, susanne
<srohrerNOsrSPAM at immv.unizh.ch.invalid> wrote:

> > If you want to transfer large bands you should treat gel
> > (especialy thick
> > ones) with 0.05N NaOH (1ml 10N +200ml H2O) for 15 minutes exactly
> > prior to a
> > few water rinses, pyrex exchange, 20XSSC 45mins with a change in
> > the middle.
> > If your blotting was good, a picture of the "pancake" will show
> > nearly no
> > ribosomal RNA.
> 
> Are you sure Alkali treatment is a good idea? I have switched to
> neutral blotting because I felt my larger transcripts were being
> degraded.

I haven't done alkali blotting in years. Generally, I get my best results
using 20X SSC as my transfer buffer when blotting RNA.  That's usually
what I recommend, after which I usually rinse the blot in 2x SSC and fix
the RNA by UV-crosslinking. When I look at the agarose gel afterwards,
there's rarely any trace of ribosomal RNA bands left. Usually the transfer
is complete.

Dave

-- 
Orac        |"A statement of fact cannot be insolent."--Orac
a.k.a.      |
David Gorski|"If you cannot listen to the answers, why do you
            | inconvenience me with questions?"--Orac again





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